Liver Prodrugs of Mitochondrial Proton Ionophores
20190276481 ยท 2019-09-12
Inventors
- Magnus Joakim Hansson (Landskrona, SE)
- Eskil Elmer (Lund, SE)
- Matthew Alan Gregory (Bourn, GB)
- Steven James Moss (Balsham, GB)
Cpc classification
A61K31/167
HUMAN NECESSITIES
C07F9/2441
CHEMISTRY; METALLURGY
A61P1/16
HUMAN NECESSITIES
International classification
Abstract
The present invention provides novel liver-targeted prodrugs of mitochondrial proton ionophores. These compounds have utility in medicine including their use in treatment of diseases such as NASH and NAFLD.
Claims
1: A compound of Formula (I) ##STR00060## wherein: X and X can independently be NH or O; Y is absent, CR.sub.3R.sub.4O, C(O)O, or ##STR00061## (X is phenyl substituent, Z connects to O); Y is absent, CR.sub.3R.sub.4O, C(O)O, or ##STR00062## (X is phenyl substituent, Z connects to O); Z is formula (II) Z is CHR.sub.2 (CO)OR.sub.1, Me, Et, iPr, Ph or formula (II) R.sub.1 and R.sub.1 are independently Me, Et, iPr, nPr, tBu, iBu, sBu or CH.sub.2CMe.sub.3 R.sub.2 and R.sub.2 are independently H, Me, Et, iPr, Ph, Bn R.sub.3 is H, Me, Et R.sub.4 is H, Me, Et ##STR00063## wherein: R.sub.5 is H, NO.sub.2 or ##STR00064## R.sub.6 is H, NO.sub.2, Cl, Br or I R.sub.7 is H, Me, Et, iPr, tBu, sBu, iBu, Cl, Br or I R.sub.8 is H, NO.sub.2, Cl, Br, C(CN)H(C.sub.6H.sub.4)-p-Cl R.sub.9 is H, Cl, OH or CH.sub.3 R.sub.10 is H or Cl R.sub.5 and R.sub.6 cannot both be H; when R.sub.6 is Cl, R.sub.5 cannot be H or NO.sub.2; when Z is CHR.sub.2 (CO)OR.sub.1, Me, Et, iPr then Y must be absent; when Z is CHR.sub.2 (CO)OR.sub.1 then X must be NH; when Z is Me, Et or iPr then X must be O; when Z is Formula II and R.sub.6 is NO.sub.2 then Y cannot be absent when Z is Formula II and R.sub.6 is NO.sub.2 then Y cannot be absent when Z is formula II and R.sub.6 is NO.sub.2 and Z is CHR.sub.2 (CO) OR.sub.1 then R.sub.2 and R.sub.2 cannot be H or Me; or a pharmaceutically acceptable salt thereof.
2: The A compound according to claim 1, wherein Z and/or Z are formula (II) and R.sub.5 is ##STR00065##
3: The A compound according to claim 1, wherein Z and/or Z are formula (II) and R.sub.5 is ##STR00066## and R.sub.6, R.sub.7, R.sub.8, R.sub.9 and R.sub.10 are all H.
4: The A compound according to claim 1, wherein Z and/or Z are formula (II); R.sub.5 is ##STR00067## and R.sub.6 is Cl, R.sub.7 is H or tBu, R.sub.8 is Cl and R.sub.9 is NO.sub.2, and R.sub.10 is H.
5: The A compound according to claim 1, wherein Z is CHR.sub.2 (CO) OR.sub.1 and Z is formula (II) and R.sub.5 is ##STR00068##
6: The A compound according to claim 1, wherein Z is CHR.sub.2 (CO)OR.sub.1 R.sub.1 and R.sub.1 are iPr R.sub.2 and R.sub.2 are Me or Bn Z is formula (II) R.sub.5 is ##STR00069## and R.sub.6, R.sub.7, R.sub.8, R.sub.9 and R.sub.10 are all H.
7: The compound according to claim 1, wherein Z is CHR.sub.2 (CO) OR.sub.1 R.sub.1 and R.sub.1 are iPr R.sub.2 and R.sub.2 are Me or Bn Z is formula (II) R.sub.5 is ##STR00070## and R.sub.6 is Cl, R.sub.7 is H or tBu, R.sub.8 is Cl and R.sub.9 is NO.sub.2, and R.sub.10 is H.
8: The compound according to claim 1 having one of the following formulas: ##STR00071##
9: The compound according to claim 1, wherein the compound is selected from: ##STR00072## ##STR00073## ##STR00074## ##STR00075## ##STR00076##
10: The compound according to claim 1 selected from ##STR00077##
11. (canceled)
12. (canceled)
13: A method of preventing or treating a disorder or disease where liver targeted mitochondrial uncoupling is useful, the method comprising administering to the subject an effective amount of a compound according to claim 1.
14: A method of preventing or treating a disorder or disease where liver targeted mitochondrial uncoupling is useful, the method comprising administering to the subject an effective amount of salicylanilide.
15: A pharmaceutical composition comprising a compound according to claim 1 together with one pharmaceutically acceptable excipients.
16: A method of treating a subject suffering from non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD), the method comprising administering to the subject an effective amount of a compound according to claim 1.
17: The method of claim 14, wherein said disorder or disease is non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD).
Description
LEGENDS TO FIGURES
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EXPERIMENTAL
[0127] A broad series of protonophore chemical classes were assessed for mitochondrial uncoupling activity to look for uncoupling potency in combination with low cellular toxicity. Liver-targeted prodrugs were then generated and tested in preclinical models.
[0128] Assessment of mitochondrial uncoupling activity revealed a number of classes of protonophores, which showed significantly less toxicity than DNP, but with improved uncoupling potency. A series of prodrugs were then generated with chemistry aimed to liver-target the protonophore. The prodrugs induced uncoupled mitochondrial respiration in liver cells with low micromolar potencies similar to the payload protonophores but lacked effect on isolated liver mitochondria. The therapeutic range of respiratory stimulation was widened and the maximal induced respiration was less than half compared to the payload protonophores. Compounds of the invention will be selected and investigated for impact on a number of preclinical markers of NASH and tolerability. Preclinical assessment of compounds of the invention suggests that it can cause liver-targeted mild mitochondrial uncoupling, without off-target issues associated with historical mitochondrial uncouplers, such as DNP. Preclinical assessment suggests it has potential as a treatment for NAFLD and NASH.
General Biology Methods
Measurement of Bioavailability
[0129] A person of skill in the art will be able to determine the pharmacokinetics and bioavailability of the compound of the invention using in vivo and in vitro methods known to a person of skill in the art, including but not limited to those described below and in Gallant-Haidner et al, 2000 and Trepanier et al, 1998 and references therein. This can be used to determine the relative exposure of the protonophore moiety in liver versus muscle and other organs. The bioavailability of a compound is determined by a number of factors, (e.g. water solubility, cell membrane permeability, the extent of protein binding and metabolism and stability) each of which may be determined by in vitro tests as described in the examples herein, it will be appreciated by a person of skill in the art that an improvement in one or more of these factors will lead to an improvement in the bioavailability of a compound. Alternatively, the bioavailability of the compound of the invention may be measured using in vivo methods as described in more detail below, or in the examples herein.
[0130] In order to measure bioavailability in vivo, a compound may be administered to a test animal (e.g. mouse or rat) both intraperitoneally (i.p.) or intravenously (i.v.) and orally (p.o.) and blood samples are taken at regular intervals to examine how the plasma concentration of the drug varies over time. The time course of plasma concentration over time can be used to calculate the absolute bioavailability of the compound as a percentage using standard models. An example of a typical protocol is described below.
[0131] For example, mice or rats are dosed with 1 or 3 mg/kg of the compound of the invention i.v. or 1, 5 or 10 mg/kg of the compound of the invention p.o. Blood samples are taken at 5 min, 15 min, 1 h, 4 h and 24 h intervals, and the concentration of the compound of the invention in the sample is determined via LCMS-MS. The time-course of plasma or whole blood concentrations can then be used to derive key parameters such as the area under the plasma or blood concentration-time curve (AUCwhich is directly proportional to the total amount of unchanged drug that reaches the systemic circulation), the maximum (peak) plasma or blood drug concentration, the time at which maximum plasma or blood drug concentration occurs (peak time), additional factors which are used in the accurate determination of bioavailability include: the compound's terminal half-life, total body clearance, steady-state volume of distribution and F %.
[0132] These parameters are then analysed by non-compartmental or compartmental methods to give a calculated percentage bioavailability, for an example of this type of method see Gallant-Haidner et al, 2000 and Trepanier et al, 1998, and references therein.
Efficacy Measurement
[0133] The efficacy of the compound of the invention may be tested using one or more of the methods described below:
1. Assays for Evaluating Mitochondrial Uncoupling
Assay for Evaluating Uncoupling Potential in Isolated Mitochondria
[0134] The potency of mitochondrial uncoupling without prodrug metabolism may be tested as follows:
[0135] Isolated rat liver mitochondria are prepared according to Hansson et al (Hansson et al (Brain Res. 2003 Jan. 17; 960(1-2):99-111.). Respiration is measured at a constant temperature of 37 C. in a high-resolution oxygraph (Oxygraph-2k Oroboros Instruments, Innsbruck, Austria) in 2 ml glass chambers with stirrer speed 750 rpm. Data is recorded with DatLab software (Oroboros Instruments, Innsbruck, Austria) with sampling rate set to 2 s at an oxygen concentration in the range of 210-50 M O.sub.2. If necessary, reoxygenation is performed by partially raising the chamber stopper for a brief air equilibration. Instrumental background oxygen flux is measured in a separate set of experiments and automatically corrected for in the ensuing experiments according to the manufacturer's instructions. To measure respiration of isolated mitochondria, samples are suspended in a mitochondrial respiration medium (MiR05) containing sucrose 110 mM, HEPES 20 mM, taurine 20 mM, K-lactobionate 60 mM, MgCl.sub.2 3 mM, KH.sub.2PO.sub.4 10 mM, EGTA 0.5 mM, BSA 1 g/l, pH 7.1. After reaching stabilized respiration in the presence of substrates (malate (5 mM), glutamate (5 mM), pyruvate (5 mM) and succinate (10 mM)), state 3 respiration is induced by supplementation with ADP (1 mM) followed by addition of oligomycin (1 g/ml, ATP-synthase inhibitor) causing state 4.sub.O. State 4.sub.O is a respiratory state dependent on the back-flux of protons across the mitochondrial membrane due to inhibition of the ATP-synthase and in the presence of saturating substrate concentrations and ADP. Drug candidates and their respective payloads of known protonophores are given at fixed concentrations to induce uncoupled respiration. Rotenone (2 M, complex I [CI] inhibitor), antimycin-A (1 g/ml, complex III [CIII] inhibitor) and sodium azide (10 mM) are then added to inhibit the ETS providing the residual, non-mitochondrial oxygen consumption which all respiratory values are corrected for.
2. Assays for Evaluating Mitochondrial Uncoupling in Intact Liver Cells and Platelets
[0136] For respiration measurements in HepG2 cells and platelets, cells are suspended in a mitochondrial respiration medium MiR05 at 37 C. in a high-resolution oxygraph (Oxygraph-2k Oroboros Instruments, Innsbruck, Austria). Initially, samples are left to stabilise at a routine respiration state, revealing resting cellular energy demands on oxidative phosphorylation (OXPHOS) of endogenous substrates. To evaluate the contribution of respiration independent of ADP phosphorylation, oligomycin (1 g/ml, ATP-synthase inhibitor) is sequentially added inducing LEAK respiration state (a respiratory state where oxygen consumption is dependent on the back-flux of protons across the mitochondrial membrane). Drug candidates and known protonophores are carefully titrated to induce maximal uncoupled respiration/maximal rate of the ETS (electron transport system) at endogenous substrate supply and continued until a decrease or at least no further increase of uncoupled respiration is observed. Rotenone (2 M, complex I [CI] inhibitor) and antimycin-A (1 g/ml, complex III [CIII] inhibitor) are then added to inhibit the ETS, thus providing the residual, non-mitochondrial oxygen consumption, which all values were corrected for.
[0137] The potency of mitochondrial uncoupling with prodrug metabolism be tested as follows: [0138] a) HepG2 cells (to simulate uncoupling with liver cell metabolism) [0139] b) Platelets (to simulate uncoupling in blood)
Hepatocyte Stability Assay
[0140] Cryopreserved hepatocytes, previously stored in liquid nitrogen are placed in a 371 C. shaking water bath for 2 min15 sec. The hepatocytes are then added to 1 OX volume of pre-warmed Krebs-Henseleit bicarbonate (KHB) buffer (2000 mg/L glucose, without calcium carbonate and sodium bicarbonate, Sigma), mixed gently and centrifuged at 500 rpm for 3 minutes. After centrifugation, the supernatant is carefully removed and a 10 volume of pre-warmed KHB buffer added to resuspend the cell pellet. This is mixed gently and centrifuged at 500 rpm for 3 minutes. The supernatant is then removed and discarded. The cell viability and yield are then determined by cell counts, and these values used to generate human hepatocyte suspensions to the appropriate seeding density (viable cell density=2106 cells/mL). A 2 dosing solution is prepared in pre-warmed KHB (1% DMSO) (200 M spiking solution: 20 L of substrate stock solution (10 mM) in 980 L of DMSO, 2 dosing solution: 10 L of 200 M spiking solution in 990 L of KHB (2 M after dilution). 50 L of pre-warmed 2 dosing solution is added to the wells and 50 L of pre-warmed hepatocyte solution (2106 cells/mL) added and timing started. The plate is then incubated at 37 C. 100 L of acetonitrile containing internal standard is added to each the wells after completion of incubation time (0, 15, 30, 60 and 120 minutes) mixed gently, and 50 L of pre-warmed hepatocyte solution added (2106 cells/mL). At the end of the incubation, cell viability is determined. Samples are centrifuged at 4000 rpm for 15 minutes at 4 C., supernatants diluted 2-fold with ultrapure water and compound levels analysed by LC-MS/MS.
[0141] Test compounds are prepared as stock solutions in DMSO at 10 mM concentration. The stock solutions are diluted in duplicate into PBS, pH7.4 in 1.5 mL Eppendorf tubes to a target concentration of 100 M with a final DMSO concentration of 1% (e.g. 4 L of 10 mM DMSO stock solution into 396 L 100 mM phosphate buffer). Sample tubes are then gently shaken for 4 hours at room temperature. Samples are centrifuged (10 min, 15000 rpm) to precipitate undissolved particles. Supernatants are transferred into new tubes and diluted (the dilution factor for the individual test article is confirmed by the signal level of the compound on the applied analytical instrument) with PBS. Diluted samples are then mixed with the same volume (1:1) of MeOH. Samples are finally mixed with the same volume (1:1) of ACN containing internal standard for LC-MS/MS analysis. Apparent permeability coefficient (Papp) and efflux ratio of the compound across the monolayer are calculated as follows:
[0142] The permeability coefficient (Papp) is calculated from the following equation:
[0143] Where dQ/dt is the amount of compound in basal (A-B) or apical (B-A) compartment as a function of time (nmol/s). C0 is the initial concentration in the donor (apical or basal) compartment (Mean of T=0) (nmol/mL) and A is the area of the transwell (cm.sup.2).
[0144] The efflux ratio is then calculated as:
Water Solubility Assay
[0145] Water solubility may be tested as follows: A 10 mM stock solution of the compound is prepared in 100% DMSO at room temperature. Triplicate 0.01 mL aliquots are made up to 0.5 mL with either 0.1 M PBS, pH 7.3 solution or 100% DMSO in amber vials. The resulting 0.2 mM solutions are shaken, at room temperature on an IKA vibrax VXR shaker for 6 h, followed by transfer of the resulting solutions or suspensions into 2 mL Eppendorf tubes and centrifugation for 30 min at 13200 rpm. Aliquots of the supernatant fluid are then analysed by the LCMS method as described above.
[0146] Alternatively, solubility in PBS at pH7.4 may be tested as follows: A calibration curve is generated by diluting the test compounds and control compounds to 40 M, 16 M, 4 M, 1.6 M, 0.4 M, 0.16 M, 0.04 M and 0.002 M, with 50% MeOH in H2O. The standard points are then further diluted 1:20 in MeOH:PBS 1:1. The final concentrations after 1:20 dilution are 2000 nM, 800 nM, 200 nM, 80 nM, 20 nM, 8 nM, 2 nM and 1 nM. Standards are then mixed with the same volume (1:1) of ACN containing internal standard. The samples are centrifuged (5 min, 12000 rpm), then analysed by LC/MS.
Cell Permeability Assay
Caco-2 Permeability Assay
[0147] Cell permeability may be tested as follows: The test compound is dissolved to 10 mM in DMSO and then diluted further in buffer to produce a final 10 M dosing concentration. The fluorescence marker lucifer yellow is also included to monitor membrane integrity. Test compound is then applied to the apical (A) surface of Caco-2 cell monolayers and compound permeation into the basolateral (B) compartment is measured. This is performed in the reverse direction (basolateral to apical) to investigate active transport (efflux). LC-MS/MS is used to quantify levels of both the test and standard control compounds (such as Propanolol and Acebutolol).
Materials
[0148] Unless otherwise indicated, all reagents used in the examples below are obtained from commercial sources.
EXAMPLES
[0149] Compounds of the invention were characterised by a combination of NMR spectroscopy and mass spectrometry. The examples illustrate the following compounds, but the invention is not limited thereto.
[0150] Where Formula IV is
##STR00031##
and Formula III is
[0151] ##STR00032##
TABLE-US-00001 compound R1 R2 X Y Z X Y Z 1 iPr Me O CR.sub.3R.sub.4O Formula O absent Me II 2 iPr Me O CR.sub.3R.sub.4O Formula NH absent CHR.sub.2(CO)OR.sub.1 II 3 iPr Me NH Formula III Formula O absent Me II 4 iPr Me O CR.sub.3R.sub.4O Formula NH absent CHR.sub.2(CO)OR.sub.1 II 5 iPr Me NH Formula III Formula O absent Me II 6 iPr Me O CR.sub.3R.sub.4O Formula NH absent CHR.sub.2(CO)OR.sub.1 II 7 iPr Me O CR.sub.3R.sub.4O Formula NH absent CHR.sub.2(CO)OR.sub.1 II 8 Me Me O CR.sub.3R.sub.4O Formula NH absent CHR.sub.2(CO)OR.sub.1 II 9 CH.sub.2CMe.sub.3 Me O CR.sub.3R.sub.4O Formula NH absent CHR.sub.2(CO)OR.sub.1 II 10 Et Me O CR.sub.3R.sub.4O Formula NH absent CHR.sub.2(CO)OR.sub.1 II 11 iPr Bn O CR.sub.3R.sub.4O Formula NH absent CHR.sub.2(CO)OR.sub.1 II 12 iPr Bn O CR.sub.3R.sub.4O Formula NH absent CHR.sub.2(CO)OR.sub.1 II 13 iPr Bn O CR.sub.3R.sub.4O Formula NH absent CHR.sub.2(CO)OR.sub.1 II 14 iPr Bn O CR.sub.3R.sub.4O Formula NH absent CHR.sub.2(CO)OR.sub.1 II 15 Me Me O CR.sub.3R.sub.4O Formula O absent Me II 16 iPr Me O CR.sub.3R.sub.4O Formula O absent Et II 17 iPr Me O CR.sub.3R.sub.4O Formula O absent Ph II 18 iPr Me O CR.sub.3R.sub.4O Formula O absent Me II 19 iPr Me O CR.sub.3R.sub.4O Formula O absent Me II 20 CH.sub.2CMe.sub.3 Me O CR.sub.3R.sub.4O Formula O absent Me II 21 Et Me O CR.sub.3R.sub.4O Formula O absent Me II 22 iPr Me O absent Formula NH absent CHR.sub.2(CO)OR.sub.1 II 23 CH.sub.2CMe.sub.3 Me O CR.sub.3R.sub.4O Formula NH absent CHR.sub.2(CO)OR.sub.1 II 24 iPr Me O absent Formula O absent Formula II II 25 iPr Me O CR.sub.3R.sub.4O Formula NH absent CHR.sub.2(CO)OR.sub.1 II 26 tBu iPr O CR.sub.3R.sub.4O Formula NH absent CHR.sub.2(CO)OR.sub.1 II 27 CH.sub.2CMe.sub.3 Bn O CR.sub.3R.sub.4O Formula NH absent CHR.sub.2(CO)OR.sub.1 II compound R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 1 NA NA H H NO.sub.2 NO.sub.2 H NA NA NA 2 iPr Me H H NO.sub.2 NO.sub.2 H NA NA NA 3 NA NA NA NA NO.sub.2 NO.sub.2 H NA NA NA 4 iPr Me H H H NO.sub.2 H NA NA NA 5 NA NA NA NA H NO.sub.2 H NA NA NA 6 iPr Me H H Formula IV Cl H NO.sub.2 Cl H 7 iPr Me H H Formula IV H H H H H 8 Me Me H H H NO.sub.2 H NA NA NA 9 CH.sub.2CMe.sub.3 Me H H H NO.sub.2 H NA NA NA 10 Et Me H H H NO.sub.2 H NA NA NA 11 iPr Bn H H NO.sub.2 NO.sub.2 H NA NA NA 12 iPr Bn H H H NO.sub.2 H NA NA NA 13 iPr Bn H H Formula IV Cl H NO2 Cl H 14 iPr Bn H H Formula IV H H H H H 15 NA NA H H H NO.sub.2 H NA NA NA 16 NA NA H H H NO.sub.2 H NA NA NA 17 NA NA H H H NO.sub.2 H NA NA NA 18 NA NA H H Formula IV Cl H NO2 Cl H 19 NA NA H H Formula IV H H H H H 20 NA NA H H H NO.sub.2 H NA NA NA 21 NA NA H H H NO.sub.2 H NA NA NA 22 iPr Me NA NA Formula IV H H H H H 23 CH.sub.2CMe.sub.3 Me H H Formula IV H H H H H 24 NA NA NA NA Formula IV H H H H H 25 iPr Me H H Formula IV H H H H H 26 tBu iPr H H Formula IV H H H H H 27 CH.sub.2CMe.sub.3 Bn H H Formula IV H H H H H
Example 1Compound 1
[0152] ##STR00033##
[0153] A solution of methyl phosphorodichloridate (3.0 g, 20.1 mmol) in DCM (60 ml) was added dropwise to a mixture of benzyl alcohol (2.18 g, 20.1 mmol) and triethylamine (TEA) (2.04 g, 20.1 mmol) at 0 C. under nitrogen. After addition, the reaction was stirred at room temperature for 30 min before it was re-cooled to 0 C. L-Alanine isopropyl ester hydrochloride (3.71 g, 22.2 mmol) was added to the reaction and then TEA (6.12 g, 60.4 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 2 hours before it was quenched with water. The resulting mixture was extracted with DCM twice, then the combined organic layers were dried over Na.sub.2SO.sub.4, filtered and the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to give IT-001 as a colourless oil. A mixture of IT-001 and Pd(OH).sub.2/C (100 mg) in THF (30 mL) was stirred at room temperature under hydrogen atmosphere (balloon) for 2 hours. The reaction mixture was filtered and then the solvent was removed in vacuo to give IT-002 as a colourless oil. Chloromethyl chlorosulfate (7.2 g, 43.4 mmol) was added to a mixture of 2,4-dinitrophenol (4.0 g, 21.7 mmol), tetrabutylammonium hydrogen sulfate (738 mg, 2.17 mmol) and NaHCO.sub.3 (9.2 g, 109 mmol) in DCM (80 mL) and water (80 mL) at 0 C. After addition, the mixture was stirred at room temperature overnight. The mixture was diluted with water and extracted with DCM twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo to give IT-003 as yellow oil which was used in next step without purification. A mixture of IT-002 (5.0 g, 22.2 mmol), IT-003 (4.2 g, 18.1 mmol), K.sub.2CO.sub.3 (3.75 g, 27.2 mmol) and NaI (543 mg, 3.62 mmol) in CH.sub.3CN (80 mL) was stirred at room temperature overnight. The mixture was filtered and washed with CH.sub.3CN. The solvent was removed in vacuo and the residue was purified by silica gel column chromatography and then preparative-HPLC (CH.sub.3CN/H.sub.2O) to give the title compound as slightly yellow solid.
Example 2Compound 2
[0154] ##STR00034##
[0155] A mixture of benzylalcohol (3.39 g, 31.3 mmol) and TEA (3.96 g, 39.1 mmol) was added dropwise to a solution of phosphoryl trichloride (6.0 g, 39.1 mmol) in DCM (150 mL) at 78 C. under Ar. The mixture was stirred at 78 C. for 30 min. L-alanine isopropyl ester hydrochloride (16.4 g, 97.8 mmol) was added and then TEA (19.8 g, 196 mmol) was added dropwise into the reaction mixture. After addition, the mixture was warmed to room temperature and stirred for 4 hours. The reaction mixture was quenched with water and extracted with DCM twice. The combined organic layers were dried (Na.sub.2SO.sub.4), filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (EtOAc/petrol ether) to give IT-004 as colourless oil. A mixture of IT-004 (5.0 g, 12.1 mmol) and Pd(OH).sub.2/C (1.0 g) in THF (100 mL) was stirred at room temperature under hydrogen atmosphere (balloon) for 2 hours. The reaction mixture was filtered and evaporated under reduced pressure to give IT-005 which was used for next step without purification. Chloromethyl chlorosulfate (4.0 g, 24 mmol) was added to a mixture of IT-005 (3.9 g, 12 mmol), tetrabutylammonium hydrogen sulfate (407 mg, 1.2 mmol) and NaHCO.sub.3 (6.0 g, 72 mmol) in DCM (60 mL) and water (60 mL) at room temperature and was stirred overnight. The mixture was extracted with DCM 3 times. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo to give IT-006 as slightly yellow oil which was used in next step without purification. A mixture of IT-006 (1.8 g, 4.83 mmol), 2,4-dinitrophenol (1.33 g, 7.24 mmol), K.sub.2CO.sub.3 (1.34 g, 9.66 mmol) and NaI (145 mg, 0.97 mmol) in CH.sub.3CN (27 mL) was stirred at room temperature overnight. The mixture was filtered and washed with CH.sub.3CN. The solvent was removed in vacuo and the residue was purified by silica gel column chromatography and then preparative-HPLC (CH.sub.3CN/H.sub.2O) to give the title compound as colourless oil.
Example 3Compound 3
[0156] ##STR00035##
[0157] Tert-butyldimethylsilyl chloride (2.02 g, 13.4 mmol) was added to a solution of (4-aminophenyl)methanol (1.5 g, 12.2 mmol), DMAP (491 mg, 4.02 mmol) and TEA (1.48 g, 14.6 mmol) in DMF (15 mL) at room temperature and stirred at overnight. The mixture was diluted with water and extracted with EtOAc twice. The combined organic layers were washed with brine, dried (Na.sub.2SO.sub.4), filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (EtOAc/petrol ether) to give IT-007 as light yellow oil. A solution of IT-007 (2.3 g, 9.7 mmol) and TEA (982 mg, 9.7 mmol) in DCM (5 mL) was added dropwise to a solution of methyl phosphorodichloridate (1.44 g, 9.7 mmol) in DCM (20 mL) at 78 C. under Ar. The mixture was stirred at 78 C. for 30 min before L-alanine isopropyl ester hydrochloride (1.63 g, 9.7 mmol) was added. TEA (2.45 g, 24.3 mmol) was then added dropwise into the reaction mixture. After addition, the mixture was warmed to room temperature and stirred overnight. The reaction was quenched with water and extracted with DCM twice. The combined organic layers were dried (Na.sub.2SO.sub.4), filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (EtOAc/petrol ether) to give IT-008 as a colourless oil. TBAF (1 M in THF, 6.5 mL, 6.5 mmol) was added to a solution of IT-008 (970 mg, 2.18 mmol) in THF (10 mL). The reaction was heated to 40 C. and stirred overnight then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (EtOAc/petrol ether) to give IT-009 as colourless oil. DIAD (245 mg, 1.21 mmol) was added dropwise to a solution of IT-009 (100 mg, 0.303 mmol), 2,4-dinitrophenol (111 mg, 0.606 mmol) and Ph.sub.3P (159 mg, 0.606 mmol) in THF (5 mL) at 0 C. After addition, the mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo and the residue was directly purified by preparative-TLC (EtOAc) to give the title compound as a slightly yellow solid.
Example 4Compound 4
[0158] ##STR00036##
[0159] A mixture of IT-006 (see Example 2, 1.8 g, 4.83 mmol), 4-nitrophenol (1.01 g, 7.24 mmol), K.sub.2CO.sub.3 (1.34 g, 9.66 mmol) and NaI (145 mg, 0.97 mmol) in CH.sub.3CN (27 mL) was stirred at room temperature overnight. The mixture was filtered and washed with CH.sub.3CN. The solvent was removed in vacuo and the residue was purified by silica gel column chromatography and then preparative-TLC to give the title compound as a white solid.
Example 5Compound 5
[0160] ##STR00037##
[0161] DIAD (3.91 g, 19.4 mmol) was added dropwise to a solution of IT-009 (see Example 3, 1.6 g, 4.84 mmol), 4-nitrophenol (1.01 g, 7.27 mmol) and Ph.sub.3P (2.54 g, 9.68 mmol) in THF (30 mL) at 0 C. After addition, the mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo and the residue was purified by silica gel column chromatography, preparative-HPLC (CH.sub.3CN/H.sub.2O) and preparative-TLC to give the title compound as a slightly yellow solid.
Example 6Compound 6
[0162] ##STR00038##
[0163] A mixture of IT-006 (see Example 2, 600 mg, 1.61 mmol), niclosamide (790 mg, 2.41 mmol), K.sub.2CO.sub.3 (445 mg, 3.22 mmol) and NaI (48 mg, 0.32 mmol) in CH.sub.3CN (20 mL) was stirred at room temperature overnight. The mixture was filtered and washed with CH.sub.3CN. The solvent was removed in vacuo and the residue was purified by preparative-HPLC (CH.sub.3CN/H.sub.2O) and then preparative-TLC to give the title compound as a white solid.
Example 7Compound 7
[0164] ##STR00039##
[0165] A mixture of IT-006 (see Example 2, 370 mg, 0.993 mmol), salicylanilide (317 mg, 1.49 mmol), K.sub.2CO.sub.3 (206 mg, 1.49 mmol) and NaI (30 mg, 0.2 mmol) in CH.sub.3CN (7 mL) was stirred at room temperature overnight. The mixture was filtered and washed with CH.sub.3CN. The solvent was removed and the residue was purified by silica gel column chromatography and then preparative-HPLC (CH.sub.3CN/H.sub.2O) to give the title compound as a yellow oil.
Example 8Compound 8
[0166] ##STR00040##
[0167] A mixture of benzyl alcohol (3.5 g, 32.4 mmol) and TEA (3.3 g, 32.4 mmol) was added dropwise to a solution of phosphoryl trichloride (5.0 g, 32.4 mmol) in DCM (150 mL) at 78 C. under Ar. The mixture was stirred at 78 C. for 30 min. L-alanine methyl ester hydrochloride (11.3 g, 80.9 mmol) was added and then TEA (16.4 g, 162 mmol) was added dropwise into the reaction mixture. After addition, the mixture was warmed to room temperature and stirred for 4 hours. The reaction mixture was quenched with water and extracted with DCM twice. The combined organic layers were dried (Na.sub.2SO.sub.4), filtered and the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (EtOAc/petrol ether) to give IT-010 as colourless oil. A mixture of IT-010 (1.0 g, 2.8 mmol) and Pd(OH).sub.2/C (200 mg) in THF (30 mL) was stirred at room temperature under hydrogen atmosphere (balloon) for 2 hours. The reaction mixture was filtered and evaporated under reduced pressure to give IT-011 as colourless oil which was used for next step without purification. A mixture of IT-011 (112 mg, 0.42 mmol), IT-012 (see Example 20, 118 mg, 0.63 mmol), K.sub.2CO.sub.3 (116 mg, 0.84 mmol) and NaI (13 mg, 0.084 mmol) in CH.sub.3CN (2 mL) was stirred at room temperature overnight.
[0168] The mixture was filtered and washed with CH.sub.3CN. The solvent was removed in vacuo and the residue was purified by silica gel column chromatography to give the title compound as a white solid.
Example 9Compound 9
[0169] ##STR00041##
[0170] A mixture of benzyl alcohol (571 mg, 5.28 mmol) and TEA (594 mg, 5.87 mmol) was added dropwise to a solution of phosphoryl trichloride (900 mg, 5.87 mmol) in DCM (30 mL) at 78 C. under Ar. The mixture was stirred at 78 C. for 30 min. Then a solution of IT-012 (see Example 20, 2430 mg, 15.3 mmol) and TEA (2376 mg, 23.5 mmol) in DCM (3 mL) was added dropwise into the reaction mixture. After addition, the mixture was warmed to room temperature and stirred for 4 hours. The reaction mixture was quenched with water and extracted with DCM twice. The combined organic layers were dried (Na.sub.2SO.sub.4), filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (EtOAc/petrol ether) to give IT-013 as a colourless oil. A mixture of IT-013 (1.0 g, 2.13 mmol) and Pd(OH).sub.2/C (200 mg) in THF (30 mL) was stirred at room temperature under hydrogen atmosphere (balloon) for 2 hours. The reaction mixture was filtered and evaporated under reduced pressure to give IT-014 as a colourless oil which was used for next step without purification. Chloromethyl chlorosulfate (528 mg, 3.20 mmol) was added to a mixture of IT-014 (810 mg, 2.13 mmol), tetrabutylammonium hydrogen sulfate (72 mg, 0.21 mmol) and NaHCO.sub.3 (716 mg, 8.53 mmol) in DCM (16 mL) and water (16 mL) at 5 C. After addition, the mixture was stirred at room temperature overnight. The mixture was diluted with DCM and washed with aqueous Na.sub.2CO.sub.3, water, 0.5 N HCl, water. The organic layer was dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo to give IT-015 as a colourless oil which was used in next step without purification. A mixture of IT-015 (200 mg, 0.47 mmol), 4-nitrophenol (97 mg, 0.70 mmol), K.sub.2CO.sub.3 (97 mg, 0.70 mmol) and NaI (14 mg, 0.09 mmol) in CH.sub.3CN (3 mL) was stirred at room temperature overnight. The mixture was diluted with water and extracted with EtOAc twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to give the title compound as a colourless oil.
Example 10Compound 10
[0171] ##STR00042##
[0172] A mixture of benzyl alcohol (1.9 g, 17.6 mmol) and TEA (1.98 g, 19.6 mmol) was added dropwise to a solution of phosphoryl trichloride (3.0 g, 19.6 mmol) in DCM (90 mL) at 78 C. under Ar. The mixture was stirred at 78 C. for 30 min. L-alanine ethyl ester hydrochloride (7.51 g, 48.9 mmol) was added and then TEA (11.9 g, 117 mmol) was added dropwise into the reaction mixture. After addition, the mixture was warmed to room temperature and stirred for 4 hours. The reaction mixture was quenched with water and extracted with DCM twice. The combined organic layers were dried (Na.sub.2SO.sub.4), filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (EtOAc/petrol ether) to give IT-016 as a colourless oil. A mixture of IT-016 (200 mg, 0.518 mmol) and Pd(OH).sub.2/C (40 mg) in THF (8 ml) was stirred at room temperature under hydrogen atmosphere (balloon) for 2 hours. The reaction mixture was filtered and the solvent removed in vacuo to give IT-017 as a colourless oil which was used for next step without purification. A mixture of IT-017 (154 mg, 0.52 mmol), IT-012 (see Example 20, 146 mg, 0.78 mmol), K.sub.2CO.sub.3 (143 mg, 1.04 mmol) and NaI (15 mg, 0.10 mmol) in CH.sub.3CN (2 mL) was stirred at room temperature overnight. The mixture was filtered and washed with CH.sub.3CN. The solvent was removed in vacuo and the residue was purified by silica gel column chromatography to give the title compound as an off-white solid.
Example 11Compound 11
[0173] ##STR00043##
[0174] L-phenylalanine (10 g, 60.6 mmol) was dissolved in i-PrOH (100 mL) then concentrated H.sub.2SO.sub.4 (10 mL) was added slowly. The mixture was refluxed overnight before the solvent was removed in vacuo. To the residue, ice-water was added and then the solution was basified with aqueous NaOH. The resulting mixture was extracted with DCM twice.
[0175] The combined organic layers were dried (Na.sub.2SO.sub.4), filtered and then the solvent was removed in vacuo to give IT-018 as a colourless oil which could be used in next without purification. A mixture of benzyl alcohol (1.27 g, 11.7 mmol) and TEA (1.32 g, 13.0 mmol) was added dropwise to a solution of phosphoryl trichloride (2.0 g, 13.0 mmol) in DCM (60 mL) at 78 C. under Ar. The mixture was stirred at 78 C. for 30 min. Then a solution of IT-018 (6.76 g, 32.6 mmol) and TEA (5.28 g, 52.2 mmol) in DCM (5 mL) was added dropwise into the reaction mixture. After addition, the mixture was warmed to room temperature and stirred for 4 hours. The reaction mixture was quenched with water and extracted with DCM twice. The combined organic layers were dried (Na.sub.2SO.sub.4), filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (EtOAc/petrol ether) to give IT-019 as a colourless oil. A mixture of IT-019 (2.1 g, 3.71 mmol) and Pd(OH).sub.2/C (400 mg) in THF (60 mL) was stirred at room temperature under hydrogen atmosphere (balloon) for 2 hours. The reaction mixture was filtered and then the solvent was removed in vacuo to give IT-020 as a colourless oil which was used for next step without purification. Chloromethyl chlorosulfate (926 mg, 5.61 mmol) was added to a mixture of IT-020 (1.78 g, 3.74 mmol), tetrabutylammonium hydrogen sulfate (127 mg, 0.37 mmol) and NaHCO.sub.3 (1.26 g, 15.0 mmol) in DCM (30 mL) and water (30 mL) at 5 C. After addition, the mixture was stirred at room temperature overnight. The mixture was diluted with DCM and washed with aqueous Na.sub.2CO.sub.3, water, 0.5 N HCl, water. The organic layer was dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo to give IT-021 as a colourless oil which was used in next step without purification. A mixture of IT-021 (600 mg, 1.15 mmol), 2,4-dinitrophenol (316 mg, 1.72 mmol), K.sub.2CO.sub.3 (237 mg, 1.72 mmol) and NaI (34 mg, 0.23 mmol) in CH.sub.3CN (6 mL) was stirred at room temperature overnight. The mixture was diluted with water and extracted with EtOAc twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to give the title compound as yellow oil.
Example 12Compound 12
[0176] ##STR00044##
[0177] A mixture of IT-021 (see Example 11, 320 mg, 0.61 mmol), 4-nitrophenol (127 mg, 0.92 mmol), K.sub.2CO.sub.3 (126 mg, 0.92 mmol) and NaI (18 mg, 0.12 mmol) in CH.sub.3CN (6 mL) was stirred at room temperature overnight. The mixture was diluted with water and extracted with EtOAc twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to give the title compound as a colourless oil.
Example 13Compound 13
[0178] ##STR00045##
[0179] A mixture of IT-021 (see Example 11, 320 mg, 0.61 mmol), niclosamide (301 mg, 0.92 mmol), K.sub.2CO.sub.3 (126 mg, 0.92 mmol) and NaI (18 mg, 0.12 mmol) in CH.sub.3CN (6 mL) was stirred at room temperature overnight. The mixture was filtered and then the solvent was removed in vacuo. The residue was purified by preparative-HPLC (CH.sub.3CN/H.sub.2O) and the crude product was rinsed with EtOH to give pure title compound as an off-white solid.
Example 14Compound 14
[0180] ##STR00046##
[0181] A mixture of IT-021 (see Example 11, 320 mg, 0.61 mmol), salicylanilide (196 mg, 0.92 mmol), K.sub.2CO.sub.3 (126 mg, 0.92 mmol) and NaI (18 mg, 0.12 mmol) in CH.sub.3CN (6 mL) was stirred at room temperature overnight. The mixture was diluted with water and extracted with EtOAc twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to give the title compound as a yellow oil.
Example 15Compound 15
[0182] ##STR00047##
[0183] A mixture of benzyl alcohol (1.31 g, 12.1 mmol) and TEA (1.36 g, 13.4 mmol) was added dropwise to a solution of methyl phosphorodichloridate (2.0 g, 13.4 mmol) in DCM (40 mL) at 0 C. under nitrogen. After addition, the reaction was stirred at room temperature for 30 min before it was re-cooled to 0 C. L-alanine methyl ester hydrochloride (2.25 g, 16.1 mmol) was added to the reaction and then TEA (4.08 g, 40.3 mmol) was added dropwise. The reaction mixture was stirred at room temperature overnight before being quenched with water. The resulting mixture was extracted with DCM twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to give IT-022 as a colourless oil. A mixture of IT-022 (200 mg, 0.7 mmol) and Pd(OH).sub.2/C (40 mg) in THF (6 mL) was stirred at room temperature under hydrogen atmosphere (balloon) for 2 hours. The reaction mixture was filtered and the solvent was removed from the in vacuo to give IT-023 as a colourless oil. A mixture of IT-023 (69 mg, 0.35 mmol), IT-012 (see Example 20, 98 mg, 0.52 mmol), K.sub.2CO.sub.3 (96 mg, 0.7 mmol) and NaI (10.5 mg, 0.07 mmol) in CH.sub.3CN (2 mL) was stirred at room temperature overnight. The mixture was filtered and washed with CH.sub.3CN. The solvent was removed from the filtrate in vacuo and the residue was purified by silica gel column chromatography to give the title compound as a colourless oil.
Example 16Compound 16
[0184] ##STR00048##
[0185] A mixture of benzyl alcohol (1.6 g, 14.7 mmol) and TEA (2.24 g, 22.1 mmol) was added dropwise to a solution of ethyl phosphorodichloridate (3.0 g, 18.4 mmol) in DCM (50 mL) at 0 C. under nitrogen. After addition, the reaction was stirred at room temperature for 30 min before re-cooled to 0 C. L-Alanine isopropyl ester hydrochloride (3.7 g, 22.1 mmol) was added to the reaction and then TEA (5.59 g, 55.2 mmol) was added dropwise. The reaction mixture was stirred at room temperature overnight before it was quenched with water. The resulting mixture was extracted with DCM twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to give IT-024 as a colourless oil. A mixture of IT-024 (500 mg, 1.52 mmol) and Pd(OH).sub.2/C (100 mg) in THF (20 mL) was stirred at room temperature under hydrogen atmosphere (balloon) for 2 hours. The reaction mixture was filtered and then the solvent was removed from the filtrate in vacuo to give IT-025 as a colourless oil. Chloromethyl chlorosulfate (376 mg, 2.28 mmol) was added to a mixture of IT-025 (363 mg, 1.52 mmol), tetrabutylammonium hydrogen sulfate (52 mg, 0.152 mmol) and NaHCO.sub.3 (510 mg, 6.07 mmol) in DCM (10 mL) and water (10 mL) at 0 C. After addition, the mixture was stirred at room temperature overnight. The mixture was extracted with DCM twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo to give IT-026 as a colourless oil which was used in next step without purification. A mixture of IT-026, 4-nitrophenol (211 mg, 1.52 mmol), K.sub.2CO.sub.3 (315 mg, 2.28 mmol) and NaI (46 mg, 0.3 mmol) in CH.sub.3CN (5 mL) was stirred at room temperature overnight. The mixture was diluted with water and extracted with EtOAc twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to give the title compound as a slightly yellow oil.
Example 17Compound 17
[0186] ##STR00049##
[0187] A mixture of benzyl alcohol (1.23 g, 11.4 mmol) and TEA (1.73 g, 17.1 mmol) was added dropwise to a solution of phenyl phosphorodichloridate (3.0 g, 14.2 mmol) in DCM (50 mL) at 0 C. under nitrogen. After addition, the reaction was stirred at room temperature for 30 min before it was re-cooled to 0 C. L-Alanine isopropyl ester hydrochloride (2.9 g, 17.1 mmol) was added to the reaction and then TEA (4.32 g, 42.7 mmol) was added dropwise. The reaction mixture was stirred at room temperature overnight before being quenched with water. The resulting mixture was extracted with DCM twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to give IT-027 as a colourless oil. A mixture of IT-027 (1.0 g, 2.65 mmol) and Pd(OH).sub.2/C (200 mg) in THF (20 mL) was stirred at room temperature under hydrogen atmosphere (balloon) for 2 hours. The reaction mixture was filtered and then the solvent was removed from the filtrate in vacuo to give IT-028 as a colourless oil. A mixture of IT-028 (381 mg, 1.33 mmol), IT-012 (see Example 20, 1245 mg, 6.64 mmol), K.sub.2CO.sub.3 (368 mg, 2.66 mmol) and NaI (41 mg, 0.27 mmol) in CH.sub.3CN (8 mL) was stirred at room temperature overnight. The mixture was diluted with water and extracted with EtOAc twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to give the title compound as a colourless oil.
Example 18Compound 18
[0188] ##STR00050##
[0189] A mixture of benzyl alcohol (2.18 g, 20.1 mmol) and TEA (2.04 g, 20.1 mmol) was added dropwise to a solution of methyl phosphorodichloridate (3.0 g, 20.1 mmol) in DCM (60 ml) at 0 C. under nitrogen. After addition, the reaction was stirred at room temperature for 30 min before re-cooled to 0 C. L-Alanine isopropyl ester hydrochloride (3.71 g, 22.2 mmol) was added to the reaction and then TEA (6.12 g, 60.4 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 2 hours before it was quenched with water. The resulting mixture was extracted with DCM twice, then the combined organic layers were dried over Na.sub.2SO.sub.4, filtered and the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to give IT-029 as a colourless oil. A mixture of IT-029 (1.0 g, 3.17 mmol) and Pd(OH).sub.2/C (100 mg) in THF (30 mL) was stirred at room temperature under hydrogen atmosphere (balloon) for 2 hours. The reaction mixture was filtered and then the solvent was removed in vacuo to give IT-030 as a colourless oil. Chloromethyl chlorosulfate was added (3146 mg, 19.1 mmol) to a mixture of IT-030 (715 mg, 3.2 mmol), tetrabutylammonium hydrogen sulfate (109 mg, 0.32 mmol) and NaHCO.sub.3 (3023 mg, 38.1 mmol) in DCM (20 mL) and water (20 mL) at room temperature. The mixture was stirred at room temperature overnight then extracted with DCM 3 times. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo to give IT-031 as a slightly yellow oil which was used in next step without purification. A mixture of IT-031 (240 mg, 0.88 mmol), niclosamide (430 mg, 1.31 mmol), K.sub.2CO.sub.3 (363 mg, 2.63 mmol) and NaI (66 mg, 0.44 mmol) in CH.sub.3CN (15 mL) was stirred at 40 C. for 5 hours. The mixture was cooled and filtered. The solvent was removed from the filtrate in vacuo and the residue was purified by silica gel column chromatography to give the title compound as a grey solid.
Example 19Compound 19
[0190] ##STR00051##
[0191] A mixture of IT-031 (see Example 18, 300 mg, 1.09 mmol), salicylanilide (350 mg, 1.64 mmol), K.sub.2CO.sub.3 (453 mg, 3.28 mmol) and NaI (82 mg, 0.55 mmol) in CH.sub.3CN (9 mL) was stirred at room temperature overnight. The mixture was filtered and washed with EtOAc. The solvent was removed from the filtrate in vacuo and the residue was purified by silica gel column chromatography to give the title compound as a slightly yellow solid.
Example 20Compound 20
[0192] ##STR00052##
[0193] Chloromethyl chlorosulfate (10.7 g, 64.7 mmol) was added to a mixture of 4-nitrophenol (3.0 g, 21.6 mmol), tetrabutylammonium hydrogen sulfate (732 mg, 2.16 mmol) and NaHCO.sub.3 (18.1 g, 216 mmol) in DCM (90 mL) and water (90 mL) at room temperature. The mixture was stirred at 40 C. overnight. The mixture was cooled, diluted with water and extracted with DCM twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to give IT-012 as a colourless oil. A mixture of L-alanine (60.0 g, 0.673 mol), 2,2-dimethylpropan-1-ol (59.4 g, 0.673 mol) and p-toluenesulfonic acid (p-TSA) monohydrate (140.9 g, 0.741 mol) in toluene (1000 mL) was heated to reflux overnight, using a Dean-Stark apparatus. The reaction mixture was cooled and the precipitate was collected by filtration to give the product (80 g) as the p-toluenesulfonate salt. The p-toluenesulfonate salt (40 g) was dissolved in water and basified to pH=9-10 by aqueous Na.sub.2CO.sub.3. The resulting solution was extracted with DCM 3 times. The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4 and the solvent was removed in vacuo to give free IT-032 as a colourless oil. A mixture of benzyl alcohol (1.5 g, 13.9 mmol) and TEA (1.4 g, 13.9 mmol) was added dropwise to a solution of methyl phosphorodichloridate (2.1 g, 13.9 mmol) in DCM (30 ml) at 0 C. under nitrogen. After addition, the reaction was stirred at room temperature for 30 min before it was re-cooled to 0 C. A solution of IT-032 (2.4 g, 15.3 mmol) and TEA (2.1 g, 20.8 mmol) in DCM (10 mL) was added dropwise to the reaction. The reaction mixture was stirred at room temperature for 4 hours then quenched with water. The resulting mixture was extracted with DCM twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to give IT-033 as a colourless oil. A mixture of IT-033 (100 mg, 0.29 mmol) and Pd(OH).sub.2/C (20 mg) in THF (5 mL) was stirred at room temperature under hydrogen atmosphere (balloon) for 2 hours. The reaction mixture was filtered and then the solvent from the filtrate was removed in vacuo to give IT-034 as a colourless oil. A mixture of IT-034 (74 mg, 0.29 mmol), IT-012 (82 mg, 0.44 mmol), K.sub.2CO.sub.3 (81 mg, 0.58 mmol) and NaI (13 mg, 0.088 mmol) in CH.sub.3CN (2 mL) was stirred at room temperature overnight. The mixture was filtered and washed with CH.sub.3CN. The solvent from the filtrate was removed in vacuo and the residue was purified by silica gel column chromatography to give the title compound as a slightly yellow oil.
Example 21Compound 21
[0194] ##STR00053##
[0195] A mixture of benzyl alcohol (1.5 g, 13.9 mmol) and TEA (1.4 g, 13.9 mmol) was added dropwise to a solution of methyl phosphorodichloridate (2.1 g, 13.9 mmol) in DCM (30 ml) at 0 C. under nitrogen. After addition, the reaction was stirred at room temperature for 30 min then re-cooled to 0 C. Ethyl L-alaninate hydrochloride (2.35 g, 15.3 mmol) was added to the reaction and then TEA (4.2 g, 41.7 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 4 hours then quenched with water.
[0196] The resulting mixture was extracted with DCM twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to give IT-035 as a colourless oil. A mixture of IT-035 (200 mg, 0.66 mmol) and Pd(OH).sub.2/C (40 mg) in THF (8 mL) was stirred at room temperature under hydrogen atmosphere (balloon) for 2 hours. The reaction mixture was filtered and then the solvent from the filtrate was removed in vacuo to give IT-036 as a colourless oil. A mixture of IT-036 (141 mg, 0.67 mmol), IT-012 (see Example 20, 188 mg, 1.0 mmol), K.sub.2CO.sub.3 (185 mg, 1.3 mmol) and NaI (30 mg, 0.2 mmol) in CH.sub.3CN (3 mL) was stirred at room temperature overnight. The mixture was filtered and washed with CH.sub.3CN. The solvent from the filtrate was removed in vacuo and the residue was purified by silica gel column chromatography to give the title compound as a slightly yellow oil.
Example 22Compound 22
[0197] ##STR00054##
[0198] TEA (1.9 g, 18.8 mmol) was added dropwise to a solution of POCl.sub.3 (2.85 g, 18.8 mmol) and salicylanilide (4.0 g, 18.8 mmol) in dry DCM (100 ml) at 78 C. under an atmosphere of Argon. The mixture was stirred at 78 C. for 30 min. L-Alanine isopropyl ester hydrochloride (7.9 g, 46.9 mmol) was added to the reaction and then TEA (11.4 g, 112.7 mmol) was added dropwise at 78 C. The reaction mixture was stirred at room temperature for 3 hours before it was quenched with water. The resulting mixture was extracted with DCM twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography twice (EtOAc/petroleum ether=1/3 to 1/2) to give the title compound as a slightly yellow oil.
Example 23Compound 23
[0199] ##STR00055##
[0200] A mixture of phenylmethanol (571 mg, 5.28 mmol) and TEA (594 mg, 5.87 mmol) was added dropwise to a solution of phosphoryl trichloride (900 mg, 5.87 mmol) in DCM (30 mL) at 78 C. under inert conditions. The mixture was stirred at the same temperature for 30 minutes then a solution of IT-032 (see Example 20, 2430 mg, 15.3 mmol) and TEA (2376 mg, 23.5 mmol) in DCM (3 mL) was added dropwise. The mixture was then warmed to room temperature and stirred for another 4 hours. The reaction mixture was quenched with water and extracted with DCM twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (EtOAc/petrol ether) to give IT-037 as colorless oil. A mixture of IT-037 (1.0 g, 2.13 mmol) and Pd(OH).sub.2/C (200 mg) in THF (30 mL) was stirred at room temperature under hydrogen atmosphere (balloon) for 2 hours. The reaction mixture was filtered and the solvent evaporated under reduced pressure to give IT-038 as colorless oil which was used for next step without purification. Chloromethyl chlorosulfate (528 mg, 3.20 mmol) was added to a mixture of IT-038 (810 mg, 2.13 mmol), tetrabutylammonium hydrogen sulfate (72 mg, 0.21 mmol) and NaHCO.sub.3 (716 mg, 8.53 mmol) in DCM (16 mL) and water (16 mL) at 5 C. After addition, the mixture was stirred at room temperature overnight. The mixture was then diluted with DCM and successively washed with saturated aqueous Na.sub.2CO.sub.3 solution, water, then 0.5 N HCl and water. The organic layer was dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo to give IT-039 as colorless oil which was used in next step without purification. A mixture of IT-039 (400 mg, 0.93 mmol), 2-hydroxy-N-phenylbenzamide (298 mg, 1.40 mmol), K.sub.2CO.sub.3 (193 mg, 1.40 mmol) and NaI (28 mg, 0.18 mmol) in CH.sub.3CN (10 mL) was stirred at room temperature overnight. The mixture was diluted with water and extracted with EtOAc twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to give compound 23 as colorless oil.
Example 24Compound 24
[0201] ##STR00056##
[0202] TEA (1.2 g, 12 mmol) was added dropwise to a solution of POCl.sub.3 (912 mg, 6 mmol) and L-Alanine isopropyl ester hydrochloride (1 g, 6 mmol) in dry DCM (30 ml) at 78 C. under inert conditions. The mixture was stirred at 78 C. for 1 hour. Salicylanilide (2.6 g, 12 mmol) was then added followed by the addition of TEA (1.2 g, 12 mmol) dropwise at 78 C. The resulting mixture was stirred at room temperature for another 3 hours before it was quenched with water. The solvent was removed in vacuo and the residue was purified by prep-HPLC to give compound 24 as white solid.
Example 25Compound 25
[0203] ##STR00057##
[0204] A mixture of phenylmethanol (633 mg, 5.86 mmol) and TEA (658 mg, 6.51 mmol) was added dropwise to a solution of phosphoryl trichloride (1.0 g, 6.51 mmol) in DCM (30 mL) at 78 C. under inert conditions. The mixture was stirred at 78 C. for 30 minutes. Then a solution of (S)-isopropyl 2-aminopropanoate hydrochloride (2.73 g, 16.28 mmol) and TEA (3.4 g, 33.85 mmol) were added dropwise into the reaction mixture separately. After addition, the mixture was warmed to room temperature and stirred for 4 hours. The reaction mixture was quenched with water and extracted with DCM twice.
[0205] The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (EA/PE=1/2) to give 25-2 as colorless oil. A mixture of 25-2 (1.85 g, 4.47 mmol) and Pd(OH).sub.2/C (600 mg) in THF (30 mL) was stirred at room temperature under hydrogen atmosphere (balloon) for 2 hours. The reaction mixture was filtered and the solvent evaporated under reduced pressure to give 25-3 as colorless oil which was used for next step without purification. Chloromethyl chlorosulfate (1.16 g, 7.05 mmol) was added to a mixture of 25-3 (1.53 g, 4.7 mmol), tetrabutylammonium hydrogen sulfate (160 mg, 0.47 mmol) and NaHCO.sub.3 (1.6 g, 18.8 mmol) in DCM (20 mL) and water (20 mL) at 5 C. After addition, the mixture was stirred at room temperature overnight. The mixture was diluted with DCM and washed with aqueous Na.sub.2CO.sub.3, water, 0.5 N HCl, water. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give 25-4 as colorless oil which was used in next step without purification. A mixture of 25-4 (900 mg, 2.42 mmol), 2-hydroxy-N-phenylbenzamide (773 mg, 3.63 mmol), K.sub.2CO.sub.3 (501 mg, 1.5 mmol) and NaI (72 mg, 0.48 mmol) in CH.sub.3CN (10 mL) was stirred at room temperature overnight. The mixture was diluted with water and extracted with EtOAc twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by silica gel column chromatography (EA/PE=1/2) to give 25 as colorless oil.
Example 26Compound 26
[0206] ##STR00058##
[0207] A mixture of phenylmethanol (633 mg, 5.86 mmol) and TEA (658 mg, 6.51 mmol) was added dropwise to a solution of phosphoryl trichloride (1.0 g, 6.51 mmol) in DCM (30 mL) at 78 C. under inert conditions. The mixture was stirred at 78 C. for 30 minutes. Then a solution of (S)-tert-butyl 2-amino-3-methylbutanoate hydrochloride (3.0 g, 14.3 mmol) and TEA (3.02 g, 29.9 mmol) were separately added dropwise into the reaction mixture. After addition, the mixture was warmed to room temperature and stirred for 4 hours. The reaction mixture was quenched with water and extracted with DCM twice. The combined organic layers were dried (Na.sub.2SO.sub.4), filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (EA/PE=1/2) to give 26-2 as colorless oil. A mixture of 26-2 (2.1 g, 4.2 mmol) and Pd(OH).sub.2/C (500 mg) in THF (30 mL) was stirred at room temperature under hydrogen atmosphere (balloon) for 2 hours. The reaction mixture was filtered and evaporated under reduced pressure to give 26-3 (as colorless oil which was used for next step without purification. Chloromethyl chlorosulfate was added (1.49 mg, 9.03 mmol) to a mixture of 26-3 (2.46 g, 6.02 mmol), tetrabutylammonium hydrogen sulfate (204 mg, 0.6 mmol) and NaHCO.sub.3 (2.02 g, 24.08 mmol) in DCM (30 mL) and water (30 mL) at 5 C. After addition, the mixture was stirred at room temperature overnight. The mixture was diluted with DCM and washed with aqueous Na.sub.2CO.sub.3, water, 0.5 N HCl, water. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give 26-4 as colorless oil which was used in next step without purification. A mixture of 26-4 (2.1 g, 4.73 mmol), 2-hydroxy-N-phenylbenzamide (1.51 g, 7.1 mmol), K.sub.2CO.sub.3 (980 mg, 7.1 mmol) and NaI (142 mg, 0.95 mmol) in CH.sub.3CN (20 mL) was stirred at room temperature overnight. The mixture was diluted with water and extracted with EtOAc twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by silica gel column chromatography (EA/PE=1/2) to give 26 as colorless oil.
Example 27Compound 27
[0208] ##STR00059##
[0209] A mixture of 27-0 (3.3 g, 20 mmol), 2,2-dimethylpropan-1-ol (3.5 g, 40 mmol), and p-toluenesulfonic acid (PTSA) monohydrate (4.1 g, 24 mmol) in toluene (50 mL) was heated with a Dean-Stark trap, and kept at reflux temperature overnight. The reaction mixture was cooled and then the solvent was removed in vacuo. The residue was dissolved in DCM and basified to pH 9-10 by saturated aqueous Na.sub.2CO.sub.3. The resulting solution was extracted with DCM 3 times. The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4 and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (EtOAc/petrol ether=1/10 to 1/1) to give 27-1 as yellow oil. A mixture of phenylmethanol (380 mg, 3.52 mmol) and TEA (395 mg, 3.91 mmol) was added dropwise to a solution of phosphoryl trichloride (600 mg, 3.91 mmol) in DCM (15 mL) at 78 C. under Ar and stirred for 30 min. Separately, a solution of 27-1 (2.2 g, 9.38 mmol) in DCM (5 mL) and then TEA (1.42 g, 14.01 mmol) in DCM (5 mL) were added dropwise into the reaction mixture at 78 C. After addition, the mixture was warmed to room temperature and stirred for 4 hours. The reaction mixture was quenched with water and extracted with DCM twice. The combined organic layers were dried (Na.sub.2SO.sub.4), filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (EtOAc/petrol ether=1/10 to 1/1) to give 27-2 as a colourless oil. A mixture of 27-2 (1.3 g, 2.09 mmol) and Pd(OH).sub.2/C (300 mg) in THF (20 mL) was stirred at room temperature under hydrogen atmosphere (balloon) for 2 hours. The reaction mixture was filtered and the filtrate then the solvent was removed in vacuo to give 27-3 as a colourless oil. Chloromethyl chlorosulfate (436 mg, 2.64 mmol) was added to a mixture of 27-3 (938 mg, 1.76 mmol), tetrabutylammonium hydrogen sulfate (60 mg, 0.18 mmol) and NaHCO.sub.3 (591 mg, 7.04 mmol) in DCM (10 mL) and water (10 mL) at 5 C. After addition, the mixture was stirred at room temperature overnight. The mixture was diluted with DCM and washed with aqueous Na.sub.2CO.sub.3, water, 0.5 N HCl, water. The organic layer was dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo to give 27-4 as a colourless oil which was used in next step without purification. A mixture of 27-4 (432 mg, 0.74 mmol), 2-hydroxy-N-phenylbenzamide (238 mg, 1.11 mmol), K.sub.2CO.sub.3 (153 mg, 1.11 mmol) and NaI (22 mg, 0.15 mmol) in CH.sub.3CN (8 mL) was stirred at room temperature overnight. The mixture was diluted with water and extracted with EtOAc twice. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (EA/PE=1/10 to 1/1) to give 27 as white solid.
Example 28Analysis of Prodrug Uncoupling
[0210] A selection of compounds of the invention were tested for free mitochondrial uncoupling and compared to known potent uncouplers DNP and MNP. The results are shown in
[0211] As can be seen from the data, Compounds 1, 2, 4, 6, 9, 11, 14, 18 and 23 show reduced, little or no uncoupling in this assay, whilst DNP, MNP and niclosamide show potent uncoupling. This shows that metabolism (such as hepatic metabolism) is required for a significant uncoupling effect, allowing for improved liver targeting.
Example 29Analysis of Uncoupling Activity of Salicylanilide
[0212] Salicylanilide and DNP were compared in a mitochondrial uncoupling assay in intact HepG2 liver cells. The results are shown in
Example 30Analysis of Relative Liver Exposure Vs Extra-Hepatic Organs
[0213] As it is advantageous to have an increased ratio of liver uncoupling versus extra hepatic uncoupling, 3 mg/kg salicylanilide or 10 mg/kg compound 14 or 10 mg/kg compound 23 (which releases salicylanilide) were dosed orally to CD-1 mice and levels of salicylanilide were measured in blood, muscle and liver samples before and after dosing (see general methods). The ratio of liver vs extra-hepatic salicylanilide was then assessed, with a high ratio desirable, as this is anticipated to lead to reduced off-target uncoupling and toxicity.
TABLE-US-00002 Salicylanilide Salicylanilide Salicylanilide Ratio of Ratio of in liver after 1 h in muscle after in blood after liver to liver to Compound (ng/g) 1 h (ng/g) 1 h (ng/g) muscle blood Salicylanilide 694 7 14 99 50 Compound 14 126 4 BQL 32 N/A Compound 23 460 4 4 115 115
[0214] As can be seen from the data above, salicylanilide, compounds 14 and 23 all have desirable ratios of liver to extra-hepatic exposure.
Example 31Comparison of Extrahepatic Uncoupling Vs Hepatic Uncoupling In Vitro
[0215] It is advantageous to have an increased level of uncoupling in hepatic tissue as compared to extra-hepatic tissue. To test for this, compounds were tested in an in vitro uncoupling assay (see Assays for evaluating mitochondrial uncoupling in intact liver cells and platelets in general methods) in HepG2 cells (hepatic) vs platelets (extra-hepatic). Data is shown in
[0216] It may be advantageous to restrict the maximum level of uncoupling of a protonophore to a level lower than that of DNP, which is known to cause side effects and death at high doses, when tested on HepG2 cells.
Example 32Comparison of Permeability of Salicylanilide Vs Other Uncoupling Agents
[0217] It is advantageous to have an increased level of oral bioavailability and cellular permeability. The potential for this can be measured by a caco-2 permeability assay (see general methods). Data is show in the table below:
TABLE-US-00003 Caco-2 A-B Caco-2 B-A (Papp, (Papp, Caco-2 Compound 1 10.sup.6 cm s.sup.1) 1 10.sup.6 cm s.sup.1) Efflux Ratio Salicylanilide 39.5 33 0.8 DNP 24 27 1.1
[0218] As can be seen from the data, salicylanilide shows increased permeability and reduced efflux ratio as compared to DNP, a well-known orally bioavailable uncoupling agent.
[0219] The application of which this description and claims forms part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein.
[0220] They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation, the following claims: All references referred to in this application, including patent and patent applications, are incorporated herein by reference to the fullest extent possible.